Chronic pain management accounts for approximately US$100 billion in direct and indirect costs. Current treatments fail to address effectively this large medical problem: narcotics cause addiction and safety risks, while other types of analgesics are hampered by insufficient efficacy and side effects. Chronic pain is a progressive condition where persistent allodynia and hyperalgesia often emerge due to the presence of chronic disease (e.g. inflammation). Therefore, there is a critical need to develop novel, safe therapies that can rectify pathological mechanisms involved in chronic inflammatory pain. We propose to develop novel inhibitors of N-acylethanolamine-hydrolyzing acid amidase (NAAA) in collaboration with Dr. Piomelli (UC Irvine) who is a recognized world-leader in the field of endogenous lipid mediators including NAAA signaling. NAAA is an enzyme that serves as a key inflammatory checkpoint and regulates pain processing by degrading palmitoylethanolamine (PEA), a lipid that exerts profound analgesic and anti-inflammatory effects in a broad range of rodent models. Moreover, PEA is analgesic in clinical pain and has been used to treat effectively pain conditions in patients but PEA is metabolically labile and has poor pharmacokinetics (PK). A better approach may be to inhibit NAAA to elevate PEA levels at inflammatory and nociceptive sites, resulting in anti-inflammatory and analgesic responses but so far only a few NAAA inhibitors with poor systemic PK have been described. The discovery of new scaffolds suitable for drug optimization has been hindered by the lack of assays compatible with high-throughput screening (HTS). To overcome this challenge we are taking two approaches: (i) we developed a new NAAA assay that is compatible with HTS, and (ii) we built a 3D-homology model of NAAA that can be used to execute virtual screens and guide the design of novel inhibitors. Using this assay we ran a pilot screen of 13K diverse compounds and identified 27 novel inhibitors that block NAAA but do not inhibit similar hydrolases. Using the 3D-homology model we have shown that these inhibitors display proper interactions in the catalytic pocket. In addition, we ran a virtual screening of 2 million commercial compounds and detected additional hits with inhibitory activity. The first two aims of this proposal will focus on (Aim#1) completing the HTS of our library of 58K small molecules and (Aim#2) the characterization of all the virtual screening hits. Aim #3 will confirm activity on NAAA, mechanisms of inhibition, selectivity over other similar hydrolases, and activity in cellular assays. The last aim (Aim#4) wil test for off-target activity on other receptors and hERG, and metabolic stability in vitro. Initial SAR of prioritized scaffolds will be explored assisted by computational methods. We will prioritize the best scaffolds for lead optimization based on pharmacology, chemical, and metabolic properties. The SBIR phase II will focus on lead optimization to improve the pharmacology and PK of lead series.